1. Field of the Invention
The present invention relates to a reagent for determination of calcium and a determination method. More particularly, it relates to an improved reagent for determination of calcium and an improved determination method which permit accurate determination of calcium in a sample derived from a living body, such as a blood sample (e.g. whole blood, plasma or serum) or an aqueous liquid sample derived from a living body (e.g. cerebrospinal fluid, lymph, salivary juice or urine) and are especially useful for clinical diagnoses. Furthermore, the present invention relates to a blank-controlling agent for chelating and color-producing agent used in the determination of metal ions by the reaction of a chelating and color-producing agent with the metal ions.
2. Description of the Related Art
As a method for quantitative analysis for calcium ions in a sample, a colorimetry method using a chelating and color-producing agent is commonly practiced for clinical laboratory tests and in the general chemical analysis fields. As the chelating and color-producing agent, o-cresolphthalein complexone (o-CPC) is mainly used which has a pH value most suitable for coloration in an alkaline pH range in which its bonding to calcium for the formation of a chelate is stable, in particular, a high pH range of pH 10 or more, as disclosed in, for example, JP-A-54-36996. There is also known a reagent obtained by incorporating 8-hydroxyquinoline into Arsenazo-III used as a chelating and color-producing agent, to stabilize this agent, as disclosed in Japanese Patent Application Kohyo No. 06-505560. In addition, there is also known an integral-type multi-layer analyzing element for calcium analysis obtained by the use of Chlorophosphonazo-III as a chelating and color-producing agent and having a determination range expanded by the use of light-scattering particles, as disclosed in JP-A-06-50976. Moreover, JP-A-04-120464 discloses that calcium and magnesium can be quantified at the same time by using Chlorophosphonazo-III as a chelating and color-producing agent.
However, each of conventional reagents for calcium determination obtained by using a chelating and color-producing agent leaves something to be improved. For example, a reagent for determination obtained by using o-CPC should be improved because it has a pH of 10 or more. That is, since this reagent has a high pH, its pH is remarkably lowered at the time of use by absorption of carbonic acid gas in the air. Moreover, when the reagent is stored in the form of a solution for a long period of time, its pH is lowered, so that measured values become inaccurate in some cases. On the other hand, the reagent for calcium determination obtained by using Arsenazo-III is advantageous in having a lower pH as compared with a method using o-CPC, but Arsenazo-III is an organic arsenic compound and hence there is no denying the problem of environmental pollution caused by the disposal of the reagent. In the case of Chlorophosphonazo-III, a pH range most suitable for its color change is weakly acidic and Chlorophosphonazo-III is a reagent containing no arsenic. Therefore, Chlorophosphonazo-III is advantageous with respect to problems caused by a high pH and toxicity. The integral-type multi-layer analyzing element for calcium analysis, however, is not a liquid reagent and does not make it possible to subject a large number of samples to determination in a short time. In addition, in the simultaneous qualification of magnesium and calcium, Chlorophosphonazo-III is added to a sample to bond both magnesium and calcium thereto and cause coloration, and then EGTA is added to dissociate calcium therefrom and cause tone reduction, and calcium is quantified on the basis of the degree of the tone reduction. Since Chlorophosphonazo-III gives high blank values, it limits a range where the determination is possible.
That is, although several reagents for calcium determination obtained by using a chelating and color-producing agent have been proposed, a reagent has not yet been proposed which can solve all of the various problems, i.e., the problem of carbonic acid gas absorption caused by a high pH, the problem of use of the toxic arsenic compound, the problem of the impossibility of determination for a large number of samples in a short time, and the problem of high blank values and hence a narrow range where the determination is possible.